| 1. |
- Mononen, Mimmi, et al.
(författare)
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Trajectory mapping of human embryonic stem cell cardiogenesis reveals lineage branch points and an ISL1 progenitor-derived cardiac fibroblast lineage
- 2020
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Ingår i: Stem Cells. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1066-5099 .- 1549-4918.
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Tidskriftsartikel (refereegranskat)abstract
- A family of multipotent heart progenitors plays a central role in the generation of diverse myogenic and nonmyogenic lineages in the heart. Cardiac progenitors in particular play a significant role in lineages involved in disease, and have also emerged to be a strong therapeutic candidate. Based on this premise, we aimed to deeply characterize the progenitor stage of cardiac differentiation at a single-cell resolution. Integrated comparison with an embryonic 5-week human heart transcriptomic dataset validated lineage identities with their late stage in vitro counterparts, highlighting the relevance of an in vitro differentiation for progenitors that are developmentally too early to be accessed in vivo. We utilized trajectory mapping to elucidate progenitor lineage branching points, which are supported by RNA velocity. Nonmyogenic populations, including cardiac fibroblast-like cells and endoderm, were found, and we identified TGFBI as a candidate marker for human cardiac fibroblasts in vivo and in vitro. Both myogenic and nonmyogenic populations express ISL1, and its loss redirected myogenic progenitors into a neural-like fate. Our study provides important insights into processes during early heart development.
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| 2. |
- Xu, Jiejia, et al.
(författare)
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Genome‐wide CRISPR screen identifies ZIC2 as an essential gene that controls the cell fate of early mesodermal precursors to human heart progenitors
- 2020
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Ingår i: Stem Cells. - Stockholm : Karolinska Institutet, Dept of Cell and Molecular Biology. - 1066-5099 .- 1549-4918.
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Tidskriftsartikel (refereegranskat)abstract
- Cardiac progenitor formation is one of the earliest committed steps of human cardiogenesis and requires the cooperation of multiple gene sets governed by developmental signaling cascades. To determine the key regulators for cardiac progenitor formation, we have developed a two-stage genome-wide CRISPR-knockout screen. We mimicked the progenitor formation process by differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes, monitored by two distinct stage markers of early cardiac mesodermal formation and commitment to a multipotent heart progenitor cell fate: MESP1 and ISL1, respectively. From the screen output, we compiled a list of 15 candidate genes. After validating seven of them, we identified ZIC2 as an essential gene for cardiac progenitor formation. ZIC2 is known as a master regulator of neurogenesis. hPSCs with ZIC2 mutated still express pluripotency markers. However, their ability to differentiate into cardiomyocytes was greatly attenuated. RNA-Seq profiling of the ZIC2-mutant cells revealed that the mutants switched their cell fate alternatively to the noncardiac cell lineage. Further, single cell RNA-seq analysis showed the ZIC2 mutants affected the apelin receptor-related signaling pathway during mesoderm formation. Our results provide a new link between ZIC2 and human cardiogenesis and document the potential power of a genome-wide unbiased CRISPR-knockout screen to identify the key steps in human mesoderm precursor cell- and heart progenitor cell-fate determination during in vitro hPSC cardiogenesis.
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| 3. |
- Adler, Andrew, et al.
(författare)
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Transsynaptic tracing and its emerging use to assess graftreconstructed neural circuits
- 2020
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 38:6, s. 716-726
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Tidskriftsartikel (refereegranskat)abstract
- Fetal neural progenitor grafts have been evaluated in preclinical animal models of spinal cord injury and Parkinson’s disease for decades, but the initial reliance on primary tissue as a cell source limited the scale of their clinical translatability. With the development of robust methods to differentiate human pluripotent stem cells to specific neural subtypes, cell replacement therapy holds renewed promise to treat a variety of neurodegenerative diseases and injuries at scale. As these cell sources are evaluated in preclinical models, new transsynaptic tracing methods are making it possible to study the connectivity between host and graft neurons with greater speed and detail than was previously possible. To date, these studies have revealed that widespread, long-lasting, and anatomically-appropriate synaptic contacts are established between host and graft neurons, as well as new aspects of host-graft connectivity which may be relevant to clinical cell replacement therapy. It is not yet clear, however, whether the synaptic connectivity between graft and host neurons is as celltype specific as it is in the endogenous nervous system, or whether that connectivity is responsible for the functional efficacy of cell replacement therapy. Here, we review evidence suggesting that the new contacts established between host and graft neuronsmay indeed be cell-type specific, and how transsynaptic tracing can be used inthe future to further elucidate the mechanisms of graft-mediated functional recovery in spinal cord injury and Parkinson’s disease.
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| 4. |
- Aldskogius, Håkan, 1943-, et al.
(författare)
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Regulation of boundary cap neural crest stem cell differentiation after transplantation
- 2009
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 27:7, s. 1592-1603
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Tidskriftsartikel (refereegranskat)abstract
- Success of cell replacement therapies for neurological disorders will dependlargely on the optimization of strategies to enhance viability and control thedevelopmental fate of stem cells after transplantation. Once transplanted,stem/progenitor cells display a tendency to maintain an undifferentiatedphenotype or differentiate into inappropriate cell types. Gain and loss offunction experiments have revealed key transcription factors which drivedifferentiation of immature stem/progenitor cells toward more mature stages andeventually to full differentiation. An attractive course of action to promotesurvival and direct the differentiation of transplanted stem cells to a specific cell type would therefore be to force expression of regulatory differentiationmolecules in already transplanted stem cells, using inducible gene expressionsystems which can be controlled from the outside. Here, we explore thishypothesis by employing a tetracycline gene regulating system (Tet-On) to drivethe differentiation of boundary cap neural crest stem cells (bNCSCs) toward asensory neuron fate after transplantation. We induced the expression of the keytranscription factor Runx1 in Sox10-expressing bNCSCs. Forced expression of Runx1strongly increased transplant survival in the enriched neurotrophic environmentof the dorsal root ganglion cavity, and was sufficient to guide differentiationof bNCSCs toward a nonpeptidergic nociceptive sensory neuron phenotype both invitro and in vivo after transplantation. These findings suggest that exogenousactivation of transcription factors expression after transplantation instem/progenitor cell grafts can be a constructive approach to control theirsurvival as well as their differentiation to the desired type of cell and thatthe Tet-system is a useful tool to achieve this.
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| 5. |
- Ameri, Jacqueline, et al.
(författare)
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FGF2 Specifies hESC-Derived Definitive Endoderm into Foregut/Midgut Cell Lineages in a Concentration-Dependent Manner.
- 2010
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 28, s. 45-56
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Tidskriftsartikel (refereegranskat)abstract
- Fibroblast growth factor (FGF) signaling controls axis formation during endoderm development. Studies in lower vertebrates have demonstrated that FGF2 primarily patterns the ventral foregut endoderm into liver and lung, whereas FGF4 exhibits broad anterior-posterior and left-right patterning activities. Furthermore, an inductive role of FGF2 during dorsal pancreas formation has been shown. However, whether FGF2 plays a similar role during human endoderm development remains unknown. Here, we show that FGF2 specifies hESC-derived definitive endoderm (DE) into different foregut lineages in a dosage-dependent manner. Specifically, increasing concentrations of FGF2 inhibits hepatocyte differentiation, whereas intermediate concentration of FGF2 promotes differentiation towards a pancreatic cell fate. At high FGF2 levels specification of midgut endoderm into small intestinal progenitors is increased at the expense of PDX1+ pancreatic progenitors. High FGF2 concentrations also promote differentiation towards an anterior foregut pulmonary cell fate. Finally, by dissecting the FGF receptor intracellular pathway that regulates pancreas specification, we demonstrate for the first time to our knowledge that induction of PDX1+ pancreatic progenitors relies on FGF2-mediated activation of the MAPK signaling pathway. Altogether, these observations suggest a broader gut endodermal patterning activity of FGF2 that corresponds to what has previously been advocated for FGF4, implying a functional switch from FGF4 to FGF2 during evolution. Thus, our results provide new knowledge of how cell fate specification of human DE is controlled - facts that will be of great value for future regenerative cell therapies.
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| 6. |
- Anderson, JD, et al.
(författare)
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Comprehensive Proteomic Analysis of Mesenchymal Stem Cell Exosomes Reveals Modulation of Angiogenesis via Nuclear Factor-KappaB Signaling
- 2016
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 34:3, s. 601-613
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stem cells (MSC) are known to facilitate healing of ischemic tissue related diseases through proangiogenic secretory proteins. Recent studies further show that MSC derived exosomes function as paracrine effectors of angiogenesis, however, the identity of which components of the exosome proteome responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry, an unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSCs and MSC derived exosomes. We probed the proteome of MSCs and MSC derived exosomes from cells cultured under expansion conditions and under ischemic tissue simulated conditions to elucidate key angiogenic paracrine effectors present and potentially differentially expressed in these conditions. In total, 6,342 proteins were identified in MSCs and 1,927 proteins in MSC derived exosomes, representing to our knowledge the first time these proteomes have been probed comprehensively. Multilayered analyses identified several putative paracrine effectors of angiogenesis present in MSC exosomes and increased in expression in MSCs exposed to ischemic tissue-simulated conditions; these include platelet derived growth factor, epidermal growth factor, fibroblast growth factor, and most notably nuclear factor-kappaB (NFkB) signaling pathway proteins. NFkB signaling was identified as a key mediator of MSC exosome induced angiogenesis in endothelial cells by functional in vitro validation using a specific inhibitor. Collectively, the results of our proteomic analysis show that MSC derived exosomes contain a robust profile of angiogenic paracrine effectors, which have potential for the treatment of ischemic tissue-related diseases.
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| 7. |
- Avaliani, Natalia, et al.
(författare)
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Optogenetics reveal delayed afferent synaptogenesis on grafted human induced pluripotent stem cell-derived neural progenitors.
- 2014
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 32:12, s. 3088-3098
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Tidskriftsartikel (refereegranskat)abstract
- Reprogramming of somatic cells into pluripotency stem cell state have opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSC), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for six weeks, lt-NES cell-derived neurons displayed neuronal properties such as TTX-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the It-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time-point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, e.g. such as epilepsy. Stem Cells 2014.
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| 8. |
- Azim, Kasum, et al.
(författare)
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Persistent Wnt/β‐Catenin Signaling Determines Dorsalization of the Postnatal Subventricular Zone and Neural Stem Cell Specification into Oligodendrocytes and Glutamatergic Neurons
- 2014
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Ingår i: Stem Cells. - Durham, United States : AlphaMed Press, Inc.. - 1066-5099 .- 1549-4918. ; 32:5, s. 1301-1312
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Tidskriftsartikel (refereegranskat)abstract
- In the postnatal and adult central nervous system (CNS), the subventricular zone (SVZ) of the forebrain is the main source of neural stem cells (NSCs) that generate olfactory neurons and oligodendrocytes (OLs), the myelinating cells of the CNS. Here, we provide evidence of a primary role for canonical Wnt/β-catenin signaling in regulating NSC fate along neuronal and oligodendroglial lineages in the postnatal SVZ. Our findings demonstrate that glutamatergic neuronal precursors (NPs) and oligodendrocyte precursors (OPs) are derived strictly from the dorsal SVZ (dSVZ) microdomain under the control of Wnt/β-catenin, whereas GABAergic NPs are derived mainly from the lateral SVZ (lSVZ) microdomain independent of Wnt/β-catenin. Transcript analysis of microdissected SVZ microdomains revealed that canonical Wnt/β-catenin signaling was more pronounced in the dSVZ microdomain. This was confirmed using the β-catenin-activated Wnt-reporter mouse and by pharmacological stimulation of Wnt/β-catenin by infusion of the specific glycogen synthase kinase 3β inhibitor, AR-A014418, which profoundly increased the generation of cycling cells. In vivo genetic/pharmacological stimulation or inhibition of Wnt/β-catenin, respectively, increased and decreased the differentiation of dSVZ-NSCs into glutamatergic NPs, and had a converse effect on GABAergic NPs. Activation of Wnt/β-catenin dramatically stimulated the generation of OPs, but its inhibition had no effect, indicating other factors act in concert with Wnt/β-catenin to fine tune oligodendrogliogenesis in the postnatal dSVZ. These results demonstrate a role for Wnt/β-catenin signaling within the dorsal microdomain of the postnatal SVZ, in regulating the genesis of glutamatergic neurons and OLs.
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| 9. |
- Baris, OR, et al.
(författare)
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The mitochondrial electron transport chain is dispensable for proliferation and differentiation of epidermal progenitor cells
- 2011
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 29:9, s. 1459-1468
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Tidskriftsartikel (refereegranskat)abstract
- Tissue stem cells and germ line or embryonic stem cells were shown to have reduced oxidative metabolism, which was proposed to be an adaptive mechanism to reduce damage accumulation caused by reactive oxygen species. However, an alternate explanation is that stem cells are less dependent on specialized cytoplasmic functions compared with differentiated cells, therefore, having a high nuclear-to-cytoplasmic volume ratio and consequently a low mitochondrial content. To determine whether stem cells rely or not on mitochondrial respiration, we selectively ablated the electron transport chain in the basal layer of the epidermis, which includes the epidermal progenitor/stem cells (EPSCs). This was achieved using a loxP-flanked mitochondrial transcription factor A (Tfam) allele in conjunction with a keratin 14 Cre transgene. The epidermis of these animals (TfamEKO) showed a profound depletion of mitochondrial DNA and complete absence of respiratory chain complexes. However, despite a short lifespan due to malnutrition, epidermal development and skin barrier function were not impaired. Differentiation of epidermal layers was normal and no proliferation defect or major increase of apoptosis could be observed. In contrast, mice with an epidermal ablation of prohibitin-2, a scaffold protein in the inner mitochondrial membrane, displayed a dramatic phenotype observable already in utero, with severely impaired skin architecture and barrier function, ultimately causing death from dehydration shortly after birth. In conclusion, we here provide unequivocal evidence that EPSCs, and probably tissue stem cells in general, are independent of the mitochondrial respiratory chain, but still require a functional dynamic mitochondrial compartment.
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| 10. |
- Behnan, Jinan, et al.
(författare)
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Recruited brain tumor-derived mesenchymal stem cells contribute to brain tumor progression.
- 2014
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 32:5, s. 1110-1123
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Tidskriftsartikel (refereegranskat)abstract
- The identity of the cells that contribute to brain tumor structure and progression remains unclear. Mesenchymal stem cells (MSCs) have recently been isolated from normal mouse brain. Here, we report the infiltration of MSC-like cells into the GL261 murine glioma model. These Brain Tumor derived Mesenchymal Stem Cells (BT-MSCs) are defined with the phenotype (Lin-Sca-1+CD9+CD44+CD166+/-) and have multipotent differentiation capacity. We show that the infiltration of BT-MSCs correlates to tumor progression; furthermore, BT-MSCs increased the proliferation rate of GL261 cells in vitro. For the first time, we report that the majority of GL261 cells expressed mesenchymal phenotype under both adherent and sphere culture conditions in vitro, and that the non-MSC population is non-tumorigenic in vivo. Although the GL261 cell line expressed mesenchymal phenotype markers in vitro, most BT-MSCs are recruited cells from host origin in both wild type GL261 inoculated into GFP-transgenic mice and GL261-GFP cells inoculated into wild type mice. We show the expression of chemokine receptors CXCR4 and CXCR6 on different recruited cell populations. In vivo, the GL261 cells change marker profile, and acquire a phenotype that is more similar to cells growing in sphere culture conditions. Finally, we identify a BT-MSC population in human glioblastoma that is CD44+CD9+CD166+ both in freshly isolated and culture-expanded cells. Our data indicate that cells with MSC-like phenotype infiltrate into the tumor stroma and play an important role in tumor cell growth in vitro and in vivo. Thus we suggest that targeting BT-MSCs could be a possible strategy for treating glioblastoma patients. Stem Cells 2013.
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| 11. |
- Berglin-Enquist, Ida, et al.
(författare)
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Successful Low-Risk Hematopoietic Cell Therapy in a Mouse Model of Type 1 Gaucher Disease
- 2009
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 27:3, s. 744-752
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Tidskriftsartikel (refereegranskat)abstract
- Hematopoietic stem cell-based gene therapy offers the possibility of permanent correction for genetic disorders of the hematopoietic system. However, optimization of present protocols is required before gene therapy can be safely applied as general treatment of genetic diseases. In this study we have used a mouse model of type 1 Gaucher disease (GD) to demonstrate the feasibility of a low-risk conditioning regimen instead of standard radiation, which is associated with severe adverse effects. We first wanted to establish what level of engraftment and glucosylceramidase (GCase) activity is required to correct the pathology of the type 1 GD mouse. Our results demonstrate that a median wild-type (WT) cell engraftment of 7%, corresponding to GCase activity levels above 10 nmoles/hour and mg protein, was sufficient to reverse pathology in bone marrow and spleen in the GD mouse. Moreover, we applied nonmyeloablative doses of busulfan as a pretransplant conditioning regimen and show that even WT cell engraftment in the range of 1%-10% can confer a beneficial therapeutical outcome in this disease model. Taken together, our data provide encouraging evidence for the possibility of developing safe and efficient conditioning protocols for diseases that require only a low level of normal or gene-corrected cells for a permanent and beneficial therapeutic outcome. STEM CELLS 2009; 27: 744-752
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| 12. |
- Bergsland, M, et al.
(författare)
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Nitric oxide-induced neuronal to glial lineage fate-change depends on NRSF/REST function in neural progenitor cells
- 2014
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 32:9, s. 2539-2549
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Tidskriftsartikel (refereegranskat)abstract
- Degeneration of central nervous system tissue commonly occurs during neuroinflammatory conditions, such as multiple sclerosis and neurotrauma. During such conditions, neural stem/progenitor cell (NPC) populations have been suggested to provide new cells to degenerated areas. In the normal brain, NPCs from the subventricular zone generate neurons that settle in the olfactory bulb or striatum. However, during neuroinflammatory conditions NPCs migrate toward the site of injury to form oligodendrocytes and astrocytes, whereas newly formed neurons are less abundant. Thus, the specific NPC lineage fate decisions appear to respond to signals from the local environment. The instructive signals from inflammation have been suggested to rely on excessive levels of the free radical nitric oxide (NO), which is an essential component of the innate immune response, as NO promotes neuronal to glial cell fate conversion of differentiating rat NPCs in vitro. Here, we demonstrate that the NO-induced neuronal to glial fate conversion is dependent on the transcription factor neuron-restrictive silencing factor-1 (NRSF)/repressor element-1 silencing transcription (REST). Chromatin modification status of a number of neuronal and glial lineage restricted genes was altered upon NO-exposure. These changes coincided with gene expression alterations, demonstrating a global shift toward glial potential. Interestingly, by blocking the function of NRSF/REST, alterations in chromatin modifications were lost and the NO-induced neuronal to glial switch was suppressed. This implicates NRSF/REST as a key factor in the NPC-specific response to innate immunity and suggests a novel mechanism by which signaling from inflamed tissue promotes the formation of glial cells. Stem Cells 2014;32:2539–2549
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| 13. |
- Bigdeli, Narmin, 1974, et al.
(författare)
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Coculture of human embryonic stem cells and human articular chondrocytes results in significantly altered phenotype and improved chondrogenic differentiation.
- 2009
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 27:8, s. 1812-21
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem (hES) cells have been suggested as a cell source for the repair of cartilage lesions. Here we studied how coculture with human articular chondrocytes affects the expansion potential, morphology, expression of surface markers, and differentiation abilities of hES cells, with special regard to chondrogenic differentiation. Undifferentiated hES cells were cocultured with irradiated neonatal or adult articular chondrocytes in high-density pellet mass cultures for 14 days. Cocultured hES cells were then expanded on plastic and their differentiation potential toward the adipogenic, osteogenic, and chondrogenic lineages was compared with that of undifferentiated hES cells. The expression of different surface markers was investigated using flow cytometry and teratoma formation was studied using injection of the cells under the kidney capsule. Our results demonstrate that although hES cells have to be grown on Matrigel, the cocultured hES cells could be massively expanded on plastic with a morphology and expression of surface markers similar to mesenchymal stem cells. Coculture further resulted in a more homogenous pellet and significantly increased cartilage matrix production, both in high-density pellet mass cultures and hyaluronan-based scaffolds. Moreover, cocultured cells formed colonies in agarose suspension culture, also demonstrating differentiation toward chondroprogenitor cells, whereas no colonies were detected in the hES cell cultures. Coculture further resulted in a significantly decreased osteogenic potential. No teratoma formation was detected. Our results confirm the potential of the culture microenvironment to influence hES cell morphology, expansion potential, and differentiation abilities over several population doublings.
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| 14. |
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| 15. |
- Blanchart, A, et al.
(författare)
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UHRF1 Licensed Self-Renewal of Active Adult Neural Stem Cells
- 2018
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 36:11, s. 1736-1751
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Tidskriftsartikel (refereegranskat)abstract
- Adult neurogenesis in the brain continuously seeds new neurons throughout life, but how homeostasis of adult neural stem cells (NSCs) is maintained is incompletely understood. Here, we demonstrate that the DNA methylation adapter ubiquitin-like, containing PHD and RING finger domains-1 (UHRF1) is expressed in, and regulates proliferation of, the active but not quiescent pool of adult neural progenitor cells. Mice with a neural stem cell-specific deficiency in UHRF1 exhibit a massive depletion of neurogenesis resulting in a collapse of formation of new neurons. In the absence of UHRF1, NSCs unexpectedly remain in the cell cycle but with a 17-fold increased cell cycle length due to a failure of replication phase entry caused by promoter demethylation and derepression of Cdkn1a, which encodes the cyclin-dependent kinase inhibitor p21. UHRF1 does not affect the proportion progenitor cells active within the cell cycle but among these cells, UHRF1 is critical for licensing replication re-entry. Therefore, this study shows that a UHRF1-Cdkn1a axis is essential for the control of stem cell self-renewal and neurogenesis in the adult brain.
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| 16. |
- Blank Savukinas, Ulrika, et al.
(författare)
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The Bystander Effect : Mesenchymal Stem Cell-Mediated Lung Repair
- 2016
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 34:6, s. 1437-1444
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Forskningsöversikt (refereegranskat)abstract
- Mesenchymal stem or stromal cells (MSCs), a heterogeneous subset of adult stem/progenitor cells, have surfaced as potential therapeutic units with significant clinical benefit for a wide spectrum of disease conditions, including those affecting the lung. Although MSCs carry both self-renewal and multilineage differentiation abilities, current dogma holds that MSCs mainly contribute to tissue regeneration and repair by modulating the host tissue via secreted cues. Thus, the therapeutic benefit of MSCs is thought to derive from so called bystander effects. The regenerative mechanisms employed by MSCs in the lung include modulation of the immune system as well as promotion of epithelial and endothelial repair. Apart from secreted factors, a number of recent findings suggest that MSCs engage in mitochondrial transfer and shedding of membrane vesicles as a means to enhance tissue repair following injury. Furthermore, it is becoming increasingly clear that MSCs are an integral component of epithelial lung stem cell niches. As such, MSCs play an important role in coupling information from the environment to stem and progenitor populations, such that homeostasis can be ensured even in the face of injury. It is the aim of this review to outline the major mechanisms by which MSCs contribute to lung regeneration, synthesizing recent preclinical findings with data from clinical trials and potential for future therapy
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| 17. |
- Blixt Wojciechowski, Anita, et al.
(författare)
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Long-term survival and glial differentiation of the brain-derived precursor cell line RN33B after subretinal transplantation to adult normal rats
- 2002
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 20:2, s. 163-173
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Tidskriftsartikel (refereegranskat)abstract
- The potential use of in vitro-expanded precursor cells or cell lines in repair includes transplantation of such cells for cell replacement purposes and the activation of host cells to provide "self-repair." Recently, we have reported that cells from the brain-derived cell line RN33B (derived from the embryonic rat medullary raphe and immortalized through retroviral transduction of the temperature-sensitive mutant of the simian virus 40 ([SV40] large T-antigen) survive for at least 4 weeks, integrate, and differentiate after subretinal grafting to normal adult rats. Here, we demonstrate that grafts of these cells survive for at least 4 months after subretinal transplantation to adult, normal immunosuppressed rats. Implanted cells integrate into the retinal pigment epithelium and the inner retinal layers, and the anterior part of the optic nerve. In addition, the RN33B cells migrate within the retina, occupying the whole retina from one eccentricity to the other. A large fraction of the grafted cells differentiate into glial cells, as shown by double labeling of the reporter genes LacZ or green fluorescent protein, and several glial markers, including oligodendrocytes. However, the cells did not differentiate into retinal neurons, judging from their lack of expression of retinal neuronal phenotypic markers. A significant number of the implanted cells in the host retina were in a proliferative stage, judging from proliferative cell nuclear antigen and SV40 large T-antigen immunohistochemistry. To conclude, the cells survived, integrated, and migrated over long distances within the host. Therefore, our results may be advantageous for future design of therapeutic strategies, since such cells may have the potential of being a source of, for example, growth factor delivery in experimental models of retinal degeneration.
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| 18. |
- Blixt Wojciechowski, Anita, et al.
(författare)
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Survival and long distance migration of brain-derived precursor cells transplanted to adult rat retina
- 2004
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Ingår i: Stem Cells. - : AlphaMed Press. - 1549-4918 .- 1066-5099. ; 22:1, s. 27-38
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Tidskriftsartikel (refereegranskat)abstract
- Neural precursor cells transplanted to adult retina can integrate into the host. This is especially true when the neural precursor rat cell line RN33B is used. This cell line carries the reporter genes LacZ and green fluorescent protein (GFP). In grafted rat eyes, RN33B cells are localized from one eccentricity to the other of the host retina. In the present study, whole-mounted retinas were analyzed to obtain a more appropriate evaluation of the amount of transgene-expressing cells and the migratory capacity of these cells 3 and 8 weeks post-transplantation. Quantification was made of the number of beta-galactosidase- and GFP-expressing cells with a semiautomatized stereological cell counting system. With the same system, delineation of the distribution area of the grafted cells was also performed. At 3 weeks, 68% of the grafted eyes contained marker-expressing cells, whereas at 8 weeks only 35% of the eyes contained such cells. Counting of marker-expressing cells demonstrated a lower number of transgene-expressing cells at 3 weeks compared with 8 weeks post-transplantation. The distribution pattern of marker gene-expressing cells revealed cells occupying up to 21% at 3 weeks and up to 68% at 8 weeks of the entire host retina post-grafting. The precursor cells survived well in the adult retina although the most striking feature of the RN33B cell line was its extraordinary migratory capacity. This capability could be useful if precursor cells are used to deliver necessary genes or gene products that need to be distributed over a large diseased area.
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| 19. |
- Bosman, A, et al.
(författare)
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Perturbations of heart development and function in cardiomyocytes from human embryonic stem cells with trisomy 21
- 2015
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 33:5, s. 1434-1446
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Tidskriftsartikel (refereegranskat)abstract
- Congenital heart defects (CHD) occur in approximately 50% of patients with Down syndrome (DS); the mechanisms for this occurrence however remain unknown. In order to understand how these defects evolve in early development in DS, we focused on the earliest stages of cardiogenesis to ascertain perturbations in development leading to CHD. Using a trisomy 21 (T21) sibling human embryonic stem cell (hESC) model of DS, we show that T21-hESC display many significant differences in expression of genes and cell populations associated with mesodermal, and more notably, secondary heart field (SHF) development, in particular a reduced number of ISL1+ progenitor cells. Furthermore, we provide evidence for two candidate genes located on chromosome 21, ETS2 and ERG, whose overexpression during cardiac commitment likely account for the disruption of SHF development, as revealed by downregulation or overexpression experiments. Additionally, we uncover an abnormal electrophysiological phenotype in functional T21 cardiomyocytes, a result further supported by mRNA expression data acquired using RNA-Seq. These data, in combination, revealed a cardiomyocyte-specific phenotype in T21 cardiomyocytes, likely due to the overexpression of genes such as RYR2, NCX, and L-type Ca2+ channel. These results contribute to the understanding of the mechanisms involved in the development of CHD. Stem Cells 2015;33:1434–1446
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| 20. |
- Bottcher, M, et al.
(författare)
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Mesenchymal Stromal Cells Disrupt mTOR-Signaling and Aerobic Glycolysis During T-Cell Activation
- 2016
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 34:2, s. 516-521
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Tidskriftsartikel (refereegranskat)abstract
- Mesenchymal stromal cells (MSCs) possess numerous regenerative and immune modulating functions. Transplantation across histocompatibility barriers is feasible due to their hypo-immunogenicity. MSCs have emerged as promising tools for treating graft-versus-host disease following allogeneic stem cell transplantation. It is well established that their clinical efficacy is substantially attributed to fine-tuning of T-cell responses. At the same time, increasing evidence suggests that metabolic processes control T-cell function and fate. Here, we investigated the MSCs' impact on the metabolic framework of activated T-cells. In fact, MSCs led to mitigated mTOR signaling. This phenomenon was accompanied by a weaker glycolytic response (including glucose uptake, glycolytic rate, and upregulation of glycolytic machinery) toward T-cell activating stimuli. Notably, MSCs express indoleamine-2,3-dioxygenase (IDO), which mediates T-cell suppressive tryptophan catabolism. Our observations suggest that IDO-induced tryptophan depletion interferes with a tryptophan-sufficiency signal that promotes cellular mTOR activation. Despite an immediate suppression of T-cell responses, MSCs foster a metabolically quiescent T-cell phenotype characterized by reduced mTOR signaling and glycolysis, increased autophagy, and lower oxidative stress levels. In fact, those features have previously been shown to promote generation of long-lived memory cells and it remains to be elucidated how MSC-induced metabolic effects shape in vivo T-cell immunity.
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| 21. |
- Brederlau, Anke, 1968, et al.
(författare)
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Transplantation of human embryonic stem cell-derived cells to a rat model of Parkinson's disease: effect of in vitro differentiation on graft survival and teratoma formation.
- 2006
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 24:6, s. 1433-40
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Tidskriftsartikel (refereegranskat)abstract
- Human embryonic stem cells (hESCs) have been proposed as a source of dopamine (DA) neurons for transplantation in Parkinson's disease (PD). We have investigated the effect of in vitro predifferentiation on in vivo survival and differentiation of hESCs implanted into the 6-OHDA (6-hydroxydopamine)-lesion rat model of PD. The hESCs were cocultured with PA6 cells for 16, 20, or 23 days, leading to the in vitro differentiation into DA neurons. Grafted hESC-derived cells survived well and expressed neuronal markers. However, very few exhibited a DA neuron phenotype. Reversal of lesion-induced motor deficits was not observed. Rats grafted with hESCs predifferentiated in vitro for 16 days developed severe teratomas, whereas most rats grafted with hESCs predifferentiated for 20 and 23 days remained healthy until the end of the experiment. This indicates that prolonged in vitro differentiation of hESCs is essential for preventing formation of teratomas.
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| 22. |
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| 23. |
- Bryja, V, et al.
(författare)
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An efficient method for the derivation of mouse embryonic stem cells
- 2006
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1066-5099 .- 1549-4918. ; 24:4, s. 844-849
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Tidskriftsartikel (refereegranskat)abstract
- Mouse embryonic stem cells (mESCs) represent a unique tool for many researchers; however, the process of ESC derivation is often very inefficient and requires high specialization, training, and expertise. To circumvent these limitations, we aimed to develop a simple and efficient protocol based on the use of commercially available products. Here, we present an optimized protocol that we successfully applied to derive ESCs from several knockout mouse strains (Wnt-1, Wnt-5a, Lrp6, and parkin) with 50%–75% efficiency. The methodology is based on the use of mouse embryonic fibroblast feeders, knockout serum replacement (SR), and minimal handling of the blastocyst. In this protocol, all centrifugation steps (as well as the use of trypsin inhibitor) were avoided and replaced by an ESC medium containing fetal calf serum (FCS) after the trypsinizations. We define the potential advantages and disadvantages of using SR and FCS in individual steps of the protocol. We also characterize the ESCs for the expression of ESC markers by immunohistochemistry, Western blot, and a stem cell focused microarray. In summary, we provide a simplified and improved protocol to derive mESCs that can be useful for laboratories aiming to isolate transgenic mESCs for the first time.
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| 24. |
- Carreira, Bruno Pereira, et al.
(författare)
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Nitric Oxide Stimulates the Proliferation of Neural Stem Cells Bypassing the Epidermal Growth Factor Receptor
- 2010
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Ingår i: Stem Cells. - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 28:7, s. 1219-1230
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Tidskriftsartikel (refereegranskat)abstract
- Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC-18 (10 mu M) increased cell proliferation, whereas higher concentrations (100 mu M) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen-activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin-dependent kinase inhibitor I, p27(KIP1), allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS(-/-) mice) prevented the increase in cell proliferation observed following seizures in wild-type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus. STEM CELLS 2010:28:1219-1230
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| 25. |
- Charbord, J, et al.
(författare)
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High throughput screening for inhibitors of REST in neural derivatives of human embryonic stem cells reveals a chemical compound that promotes expression of neuronal genes
- 2013
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Ingår i: Stem cells (Dayton, Ohio). - : Oxford University Press (OUP). - 1549-4918 .- 1066-5099. ; 31:9, s. 1816-1828
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Tidskriftsartikel (refereegranskat)abstract
- Decreased expression of neuronal genes such as brain-derived neurotrophic factor (BDNF) is associated with several neurological disorders. One molecular mechanism associated with Huntington disease (HD) is a discrete increase in the nuclear activity of the transcriptional repressor REST/NRSF binding to repressor element-1 (RE1) sequences. High-throughput screening of a library of 6,984 compounds with luciferase-assay measuring REST activity in neural derivatives of human embryonic stem cells led to identify two benzoimidazole-5-carboxamide derivatives that inhibited REST silencing in a RE1-dependent manner. The most potent compound, X5050, targeted REST degradation, but neither REST expression, RNA splicing nor binding to RE1 sequence. Differential transcriptomic analysis revealed the upregulation of neuronal genes targeted by REST in wild-type neural cells treated with X5050. This activity was confirmed in neural cells produced from human induced pluripotent stem cells derived from a HD patient. Acute intraventricular delivery of X5050 increased the expressions of BDNF and several other REST-regulated genes in the prefrontal cortex of mice with quinolinate-induced striatal lesions. This study demonstrates that the use of pluripotent stem cell derivatives can represent a crucial step toward the identification of pharmacological compounds with therapeutic potential in neurological affections involving decreased expression of neuronal genes associated to increased REST activity, such as Huntington disease.
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